ProjectSummary/Abstract Currentanti-HIV-1therapiespreventprogressiontoAIDSbutdonotcureinfection.HIV-1persistsinlong-lived memoryCD4+Tcellsasatranscriptionallysilentprovirus,whereitisundetectablebytheimmunesystem,and therefore resistant to extirpation. Recently, we reported that primate immunodeficiency virus Vpx and Vpr proteins activate HIV-1 provirus transcription by degrading the three proteins of the human silencing hub (HUSH) complex. Disruption of the HUSH complex in bulk CD4+ T cells increased transcription from HIV-1 provirusesandkineticsofHIV-1spreadinginfections,indicatingthattheHUSHcomplexplaysadominantrole inHIV-1provirussilencing.Nonetheless,examinationofindividualclonesshowedheterogeneityinresponseto HUSHdisruption,andinconsistentcorrelationwithknownsilencingfactorssuchasSETDB1.Aim1willbeto identify requirements for HUSH complex silencing of the HIV-1 provirus. Sets of CD4+ T cell clones bearing HIV-1 proviruses that exhibit a range of HUSH responsiveness will be subjected to loss-of-function screenstoidentifyhostsilencingfactorsthatdistinguishcloneswithdifferentHUSHphenotypes.Suchfactors willbecharacterizedindependentlyforeffectsonprovirustranscriptionandproviruschromatinfeatures.From these experiments we expect to better understand how HUSH is recruited to, and maintains transcriptional silencing of, HIV-1 proviruses. Aim 2 will be to examine the role of the HUSH complex in CD4+ T cell transcriptionanddevelopment.DisruptionoftheHUSHcomplexactivatesLINE-1expressionincertaincell lines raising questions about possible consequences of HUSH complex disruption. Global transcription and chromatinprofilingwillbeperformedonprimaryhumanCD4+TcellsinwhichHUSHcomplexcomponentsare disrupted. Increased expression of particular retrotransposons is expected, but also immune-related genes of relevance to HIV-1, and markers that may be used to monitor HUSH complex activity in cells. Examination of transcriptionfactormotifswithinlostATAC-SeqpeakswillaididentificationofDNA-bindingproteinsthatrecruit theHUSHcomplex.TheHUSHcomplexwillalsobedisruptedincordbloodhumanCD34+hematopoieticstem cells used to reconstitute an immune system in mice. These experiments will tell us whether the HUSH complex is essential for human hematopoietic development generally or for CD4+ T cells specifically. Aim 3 will assess the contribution of the HUSH complex to HIV-1 latency in vivo. The effect of HUSH complex inactivation on HIV-1 provirus reactivation will be examined with CD4+ T cells harvested from HIV-1+ individuals on anti-HIV-1 suppressive therapy and from humanized mice. These studies are expected to improve mechanistic understanding of HIV-1 transcriptional regulation, help prognosticate the transcriptional status of a given provirus, develop new approaches for disrupting the HIV-1 provirus in the clinical context, and,moregenerally,increasefundamentalunderstandingofgeneregulation.